Dihydrofolate reductase (DHFR) is an enzyme which is essential for cell survival and thus its gene is constitutively expressed. This enzyme is inhibited by the cancer chemotherapeutic agent, methotrexate. Cell lines, derived from human breast carcinomas have been obtained which are resistant to methotrexate. Amplification of the DHFR gene has been demonstrated in these cells by Southern blot analysis and such cells have been shown to contain an increased concentration of the mRNA for DHFR. A portion of the normal DHFR gene has been obtained as a clone from a human genomic DNA library of recombinant bacteriophage. Mapping of the 14.5 kb DNA fragment in this clone as to the location of DHFR coding sequences has been completed and the mucleotide sequence of relevant portions are being determined. A genomic DNA library has also been constructed using DNA derived from one of the methotrexate resistant cell lines.